Field of the Invention
Methods are disclosed for screening mechanical stress responses in cells and tissues using induced cavitation bubbles.
Description of the Related Art
Mechanotransduction, which refers to the mechanical forces resulting from cell-cell and cell-matrix interactions, are known to influence the signaling, function, homeostasis, and fate of individual cells and cell populations. Likewise, numerous studies have demonstrated the important role of mechanotransduction in many vital processes, including, for example: tissue morphogenesis, stem cell differentiation, vascular regulation, and tumor metastasis. Moreover, studies suggest that disruptive mechanical cues and/or dysregulation of physiological mechanotransduction pathways play important roles in the initiation and progression of numerous diseases, including, for example: atrial fibrillation, hypertension, osteoporosis, digestive diseases, and cancer. This has spurred vigorous efforts to discover therapeutic molecules that modulate cellular mechanotransduction activity, which in turn has created the need to develop assays that evaluate the sensitivity of candidate drug targets to mechanosignaling.
Currently, there are several established, high-throughput methods to precisely measure changes in cellular activity, including imaging cytometry and gene arrays. However, precise mechanical stimulation of cells can require specialized techniques such as atomic force microscopy (AFM), optical/magnetic tweezers, dynamically-stretched substrates or laminar flow chambers. While these methods are well-suited for applying physiological forces to cells in two-dimensional (2-D) and three-dimensional (3-D) cell cultures, they are time-intensive and require considerable technical expertise. Some of these methods, including optical tweezers and AFM, have utility in the study of mechanotransduction, but their low throughput means that they can only be used to examine a very limited number of cells per day. Other approaches involve the incubation of cells in specialized microdevices such as laminar flow chambers16, microfluidic chambers, and micro-fabricated substrates that expose cells to specific physiological mechanical stimuli. While such systems are well-suited to screen a small number of molecules shown to affect mechanotransduction, there is no clear path to scaling up such systems for screening hundreds of thousands of molecules.
As described above, current methods are not standardized and they are not compatible with existing high-throughput drug discovery platforms. Thus, the technical challenge involved in developing a high-throughput platform for applying precise forces to multiple cells in various culture conditions is considerable. The development of such a platform is essential to examining the role of mechanotransduction in important biological processes, and to discovering and characterizing the effects of small molecules that modulate the activity of mechano-sensitive pathways. Therefore, a practical method for the upstream, high-content screening and identification of test compounds is necessary to facilitate the discovery of a class of “mechano-active” drugs that target these “mechano-sensitive” pathways.